Project Summary Organoids are 3D mini?tissue structures that are revolutionizing in vitro studies. They can be derived from a variety of species, using stem cells or induced pluripotent stem cells (iPSCs) isolated from either normal or diseased tissues. The use of organoids in disease modeling has become a powerful method to replicate pathophysiology using relatively standard cell culture methods. Virtually every tissue type now has an in vitro organoid correlate. As the name implies, organoids are representations of tissue layers (typically epithelium) that have a specific function in an organism. They are often stem cell derived, giving them the potential to produce all the differentiated cell types found in a given tissue. While this flexibility allows recapitulation of in vivo multicellular structures, it also necessitates the use of challenging and manual cell biology protocols to establish and maintain various types of organoid. Roadblocks to rapidly establishing organoid cultures are largely due to the low efficiency of initiating a clonal colony from a single cell, difficulty tracking colony growth over time and inefficient induction of differentiation processes. In addition, systems supporting organoid culture must allow for time?course based analysis of physiological phenotypes. Cell Microsystems has developed the CellRaft Technology, a microwell array?based platform where single cells can be seeded in small culture chambers, grown into clonal colonies and tracked over time using virtually any imaging modality. During our Phase I program, we collaborated with Scott Magness, PhD of the University of North Carolina at Chapel Hill to develop organoid culture, sorting and subcloning methods using the CellRaft Technology. CytoSort Arrays, which feature thousands of microwells for cell culture, were employed to establish stem cell? derived organoid cultures. The use of Matrigel on the arrays allowed for three?dimensional support of organoid structures as they form single cells. Dr. Magness' lab also attempted organoid subcloning by isolating a single organoid from the CytoSort Array and re?plating the cells after dissociation to form second?generation organoids. In Phase II, we will leverage this powerful method on CytoSort Arrays to propagate multiple generations of organoids from a founder and undertake molecular analysis to identify lineage properties, cell types and potential accumulation of mutations leading to various disease, including cancer. To support these efforts, Cell Microsystems has developed the 3D CytoSort Array, featuring larger microwells to enable three? dimensional culture and isolation of individual organoids for molecular analysis. In Phase II we will develop protocols for other organoid types including neural, pancreatic and hepatic, optimize the overall workflow with the 3D CytoSort Array, as well as evaluate RNA?Seq analysis of multiple organoid generations. These methods are broadly applicable to organoid research in many cell and tissue types and fill an unmet need for automation in organoid workflows while retaining broad compatibility with contemporary molecular analysis methods.